Project Summary/Abstract Throughout the eukaryotic lineage, the small regulatory RNA pathway centered on PIWI (P- element-Induced WImpy testis) proteins and their associating 24~30 nucleotides (nt) PIWI-interacting RNAs (piRNAs) silence transposons and other selfish genetic elements to maintain genome integrity. PIWI proteins and piRNAs are expressed predominantly in the germline and essential for germline development. However, in contrast to other small regulatory RNA pathways, we lack a mechanistic understanding of this genome defensive system. The piRNA biogenesis how precursor RNAs are generated, processed and matured are largely unknown. Equally unclear is how the PIWI/piRNA complexes silence transposons. Our research goal is to clarify the biogenesis and function of piRNAs, and to uncover the molecular mechanism of the PIWI/piRNA pathway-mediated genome defensive system in the germline. Since recent reports have shown the human PIWI expression in various tumors, our research will impact biomedical goals of understanding diseases related to reproduction disorders and cancers. Toward the goal, we have previously discovered that PIWI proteins contain evolutionarily-conserved symmetrical dimethylarginines (sDMAs). Since TUDOR-domains of proteins are known to specifically bind to sDMAs, our discovery of PIWI sDMAs lead to our hypothesis that the important function of PIWI sDMAs is to be recognized by TUDOR-domain containing proteins, and the PIWI-TUDOR interactions play important roles in piRNA biogenesis and function. To elucidate the function of PIWI sDMAs in the piRNA pathway, we will express PIWI proteins containing or lacking sDMAs in BmN4 (a Bombyx mori ovary-derived cultured cell line) that is an excellent cell system for piRNA research due to its endogenous expression of the PIWI/piRNA pathway and convenience to use plasmid transfection and RNAi. Comparison of localization, interacting proteins, and quantity and quality of the associated piRNAs between the PIWI proteins will clarify the role of the PIWI sDMAs and their interacting TUDOR-domain containing proteins in piRNA biogenesis and function. Moreover, by systematical screening for the Bombyx TUDOR-domain containing proteins, we have recently identified BmPAPI as the factor responsible for piRNA biogenesis. We will perform biochemical, structural and functional characterizations on BmPAPI protein to investigate the molecular basis of BmPAPI-involved piRNA biogenesis. In addition, we recently found that cell-cell contact globally activates the piRNA biogenesis in BmN4 cell system. We propose to utilize this activation system to observe how piRNA clusters are transcribed to precursor RNAs and how the precursors are processed into mature piRNAs. .